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Zusammenfassung Kinetik der Enzymmuster im Hoden Basierend auf der Stadieneinteilung von Leblond und Clermont (1952a, b) wird die “Morphologie” der Enzymaktivitaten der Thiaminpyrophosphatase (TPP-ase), der sauren Phosphatasen (ACP-asen), der Adenosintriphosphatase (ATP-ase) und der Steroid-3β-ol-dehydrogenase (St-3β-ol-DH) wahrend der Spermatogenese der Ratte beschrieben. Es wird gezeigt (Fig. 1, 2) das 1 die Kinetik des Enzymmusters mit den Entwicklungsschritten wahrend der Spermatocyto- und Spermiogenese korreliert ist und damit die Chemocytoarchitektur, vor allem der Geschlechtszellen, charakteristischen Veranderungen unterworfen ist. 2 das Enzymmuster Einblicke in die Chemohistoarchitektur des Testis ermoglicht und damit auf Interaktionen zwischen Geschlechtszellen und zugeordneten somatischen Zellen hinweist. Die Kinetik des Enzymmusters last sich besonders gut am Beispiel der TPP-ase und der ACP-asen demonstrieren. Zum Zeitpunkt des ersten Auftretens — bei der TPP-ase das Praleptotanstadium in Stadium VII, bei den ACP-asen das Pachytanstadium in Stadium VII — ist die Aktivitat beider Enzyme zunachst im Cytoplasma verteilt. Unter standiger Zunahme der Aktivitat in den folgenden Stadien kommt es allmahlich zu einer kernnahen Konzentration beider Enzyme, die schlieslich in Form dichter Farbstoffkugeln im Bereich des Golgiapparates nachweisbar sind. Mit dem Eintritt in die erste und zweite Reifeteilung verschwindet die Aktivitat beider Enzyme. Nur im Golgifeldbereich der II-Spermatocyten in Interphase sind beide Enzyme schwach positiv. Mit dem Auftreten der ersten Spermatiden in Stadium I sind auch die TPP-ase und die ACP-asen wieder deutlich positiv. Im Bereich des Acrosoms finden sich Farbstoffkugeln, die deutlich kleiner sind als die Farbstoffkugeln im Bereich des Golgifeldes der Spermatocyten. In Stadium V verschwinden die ACP-asen, wahrend die TPP-ase sich in Stadium VII mit den Golgifeldern vom Acrosombereich lost und in die Schwanzregion wandert, wo sie bis zum Stadium XIV schwach nachweisbar ist. Im letzten Abschnitt der Spermiogenese, in dem die Spermatiden elongiert sind und einen besonders engen Kontakt zum Cytoplasma der Sertolizellen aufweisen, erscheinen die ACP-asen im Cytoplasma der Sertolizellen in den Stadien XII—XIV als diffuser Niederschlag im Bereich der Grenzlinie zwischen Sertolizellen und den Kopfen der elongierten Spermatiden, wahrend die TPP-ase zunachst in den Stadien XI-XIV im basalen Bereich der Sertolizellen nachweisbar ist und dann in den Stadien I—IV als wimpelformige Bander von der Basalmembran bis in die Nahe der Kopfe der elongierten Spermatiden reicht. Die ACP-asen und die TPP-ase verschwinden allmahlich in den Stadien I—III bzw. IV—V. In geringerem Mas zeigt auch die ATP-ase ein stadienabhangiges Verhalten. Von den Stadien IX—X an ist sie im apicalen Cytoplasmabereich der Sertolizellen um die Kopfe und Mittelstucke der elongierten Spermatiden nachweisbar. Sie nimmt dann an Konzentration und Kondensation bis zur Ausschleusung der Spermatozoen in die Kanalchen in Stadium VIII standig zu. Stark positiv ist die ATP-ase vor allem im boundary tissue und in der Wand der kleinen Gefase. Die St-3β-ol-DH ist innerhalb der Tubuli seminiferi nur im Bereich der elongierten Spermatiden nachweisbar. Sie ist hier schwach positiv. Die Leydigzellen zeigen eine masige bis starke Aktivitat.

The influence of cyproterone acetate (CA) upon the behaviour of hydroxysteroid dehydrogenases (HSDH) in the Wistar rat Leydig cells was investigated during the perinatal phase with the help of enzymhistochemical cum morphometrical techniques. The pregnant rats as well as their offsprings were injected with CA (dosage: 35 mg/kg body wt) sc, daily from 14 fetal day upto 31 postnatal day (p.n.d.). The animals were killed on 5, 20, and 32 p.n.d.; the enzymhistochemical reactions for 3-beta-HSDH, 11-beta-HSDH, 17-HSDH and 3-alpha-HSDH were performed in the cryostat sections of the testis, and the morphometric evaluation of HSDH positive Leydig cells was carried out. On 5 p.n.d. the activity of 17-beta-HSDH was slightly impaired in the intertubular Leydig cells of the CA treated animals. On 20 p.n.d., CA prevented nearly completely the HSDH activity in the newsly built peritubular Leydig cells; the activities of 3-beta-HSDH, 17-beta-HSDH, and 3-alpha-HSDH resided mainly in the intertubular Leydig cells. On 32 p.n.d. the HSDH activities in the Leydig cells were observed in the control as well as in treated animals. It seems that the differentiation of peritubular Leydig cells, and thereby the steroid production, is delayed by CA, but not entirely blocked.

Zusammenfassung Enzymhistochemische und morphometrische Untersuchungen an der Hydroxysteroid-Dehydrogenase wahrend der fetalen und neonatalen Entwicklung der Leydig-Zellen bei der Ratte Ziel der vorliegenden Arbeit war es, mit Hilfe der Reaktion des Enzyms Steroid-3s-ol-Dehydrogenase (3s-HSDH) quantitative Aussagen uber die Grose der „aktiven” Leydigzell-areale des intertubularen Systems zu machen und die Kinetik dieser Areale zwischen dem 17. Fetaltag und dem 5. Lebenstag zu beschreiben. Das Untersuchungsmaterial bestand aus den Gonaden mannlicher Rattenfeten vom 17. Fetaltag bis zur Geburt und von Jungtieren des 1. bis 5. Lebenstages. Die Kryostatschnitte der Testes wurden enzymhistochemisch aufgearbeitet und die Reaktion der 3s-HSDH wurde mit Dehydroepiandrosteron als Substrat durchgefuhrt. Die Areale der Leydigzellen, die eine Enzymaktivitat zeigten, wurden mor-phometrisch gemessen. 3s-HSDH-aktive Areale des intertubularen Leydigzellsystems treten am 17. Fetaltag auf. Am 19. Fetaltag erreichen die Leydigzellkomplexe im Verhaltnis zum Gesamttestis ein Maximum. Nach dem Wendepunkt des 19. Fetaltages geht der Prozentsatz des Leydigzell-systems am Gesamttestis kontinuierlich zuruck bis zum 4. Lebenstag. Das intertubulare Leydigzellsystem ist wahrend des Beobachtungsraumes aktiv und bildet sich nicht nach der Geburt zuruck. Die Grosenverteilung der Leydigzellareale andert sich wahrend des Beob-achtungszeitraumes: die grosen Komplexe weichen im Laufe der Entwicklung auseinander und bilden viele kleinere Leydigzellareale.

Summary: The aim of the present report was to study the zinc localization in rat testis with the help of a modified selective histochemical method (dithizone method). To achieve a selective demonstration of zinc, 16 μm thick serial cryostat sections of the Wistar rat testis were incubated with dithizone solution followed by a treatment with a “complexing buffer”. PAS-reaction with haematoxylin was performed to determine the stages of spermatogenesis. During the maturation phase (from stage III onwards) the red coloured zinc dithizonate granules are observed in the mid piece and tail of elongated spermatids; the number and the stain intensity of zinc granules increase continuously till the stage VII. During the stages IX–X the zinc granules, present in the residual bodies, migrate from the adluminal to the basal Sertoli cell compartment. Thereafter, the zinc granules are observed only in the boundary tissue till the stage XIV. The results lead to suggest that the residual bodies play an essential role in the zinc transport. Zusammenfassung: Zinktransport durch „Residualbodies” im Rattentestis: Eine histochemische Studie Ziel dieser Studie war es, die Zinklokalisation im Rattentestis mit Hilfe einer modifizierten, selektiven histochemischen Methode (Dithizon-Methode) zu untersuchen. 16 μm dicke Kryostatserienschnitte von Hoden der Wistarratte wurden mit einer Dithizonlosung inkubiert und anschliesend mit einem „Puffertarngemisch” behandelt, um so eine selektive Darstellung des Zinks zu erzielen. Zur Bestimmung der Spermatogenesestadien wurde die PJS-Reaktion mit Haemalaun Gegenfarbung angewandt. Die rotge-farbten Zink-Dithizon-Granula sind wahrend der Reifungsphase im Mittelstuck und im Flagellum der elongierten Spermatiden zu beobachten (ab Stadium III); die Anzahl und die Farbintensitat der Zinkgranula nehmen bis Stadium VII kontinuierlich zu. In den Stadien IX–X wandern die sich in den Residualbodies befindlichen Zinkgranula vom adluminalen zum basalen Sertolizellcompartment. Wahrend der Stadien XI–XIV beobachtet man die Zinkgranula nur im boundary tissue. Die Befunde fuhren zur Annahme, das die Residualbodies beim Zinktransport eine essentielle Rolle spielen.

The present paper is based on the knowledge of the chemo-cyto-architecture of the human testis as determined by enzyme histochemical methods. Several papers (Goslar et al., 1982; Passia et al., 1983; Passia et al., 1985) have been published on the issue of normally functioning testicular tissue as well as that of patients with disturbed fertility. All these examinations were carried out in material from young men. The question was, whether there do exist differences in the enzyme histochemical reactions of the testicular tissue in older patients as compared to young men.

The antigen CD15, also known as 3-fucosyl-N-acetyl-lactosamine (FAL), or Stage Specific Embryonic Antigen (SSEA-1) is a cell membrane and cytoplasmic antigen, that is present in several mammalian tissues. Using immunohistochemistry the monoclonal antibodies 3B9 and B4,3 detected CD15 in whole mount preparations of trophotaeniae of embryos of the matotroph viviparous teleost Xenotoca eiseni. A specific localization is encountered in the ecto- and entodermal portion of the trophotaenial epithelium. A positive reaction in the fibrocytes of the inner mesenchymal core of trophotaeniae is doubtful since a relative high background is present in these cells. CD15 positivity has been observed in all developmental stages studied. The exact role of CD15 is unclear. An involvement in endocytic activity is postulated.

Alkaline phosphatases have been demonstrated enzymhistochemically in the testis of the common frog Rana temporaria, caught in the month of January. The follicle cells in the cyst walls and the lamina propria of the seminiferous tubules showed strong enzymatic activity; also a weak activity was observed in the peripheral region of some of the germinal cells lying within the cysts. The possible interaction of alkaline phosphatases in the transport processes across the cyst walls has been discussed.

Activity of NADP+-dependent isocitrate dehydrogenase (ICDH) of the white dorsal muscle of Idus idus L. was determined by special analysis, under optimum homogenisation, centrifugation and cuvette conditions. The experimental conditions differed considerably from those used for the investigation of NADP+-ICDH activity in mammals by Bucher et al. (1964). Daily measurements of activity over a period of 10 days revealed variations in enzyme activity which are difficult to explain. However, the variations are smaller than those established in earlier tests. In order to obtain statistically valid results, the highest possible number of test fishes (at least 10 individuals for each measurement) must be investigated over a period of 5 to 10 days. NADP+-ICDH of individuals acclimated to 10°C showed 28% higher specific and non-specific activity than that of fish maintained at 20°C (experimental temperature 25°C). At 5 different adaptation temperatures, increasing activities were observed with decreasing adaptation temperature (compensation). These measurements were made over a period of 5 successive days.

Maximum substrate and cosubstrate affinity, as judged by the Michaelis constant (K M ), of NADP+-dependent isocitrate dehydrogenase of pig heart (purchased from Boehringer, Mannheim, FRG) is attained at 37°C. If K M -values of substrate (Isocitrate, IC) and cosubstrate (NADP+) of NADP+-dependent isocitrate dehydrogenase (ICDH) of the white dorsal muscle of Idus idus L. is plotted against the experimental temperature (VT), W-shaped curves result. With increasing adaptation temperature (AT), there is a shift to increasing VT. It is suggested that the W-shaped curves are due either to the simultaneous presence of two multiple forms of the enzyme, or to the reversible temperature-dependent interconversion of one protein species.

The influence of experimental thiamine deficiency in rats on enzyme-histochemical parameters was carried out in the diencephalon. The degree of thiamine deficiency was measured by the activity coefficient of transketolase in the red blood cells. The alpha-ETK of thiamine deficient animals was 1,45 +/- 0,36, of equal-fed animals 1,04 +/- 0.04 and of control animals 1,08 +/- 0,04. We got the following results: Lactatdehydrogenase, acid phosphatases and thiamine pyrophosphatase did not indicate any difference in enzyme activity between the groups of control, equal-fed and thiamine deficient rats. However, the enzyme activity of nonspecific lysosome-bound esterases in the tanycyte ependyma and in the investigated nuclear regions (nucleus supraopticus, ncl. paraventricularis, ncl. ventromedialis) of the thiamine deficient animals was significantly decreased in comparison to control and equal-fed animals. The possibility of a restriction of the hypophyseal-adrenal-cortex-system is discussed, since the results are similar to those obtained with bilaterally adrenalectomized rats.

Male C57Bl/6J-ob/ob mice (4 months old) and their homozygous lean controls were compared with respect to pituitary LH secretion and functional parameters of purified Leydig cells in vitro. Compared with controls, obese mice showed reductions in the following parameters: Plasma testosterone levels (reduced by 57%), hCG-stimulated testosterone formation in vitro (by 31%), conversion of progesterone to androgens by Leydig cells (by 39%), and GnRH-stimulated LH secretion (by 26%). Lipid accumulation and a 37% decrease in naphthylesterase activity in the Leydig cells as well as hyperplasia of pituitary gonadotrophs were observed histochemically in obese mice. The changes in testicular endocrine function in obese mice are interpreted as consequences of pituitary dysfunction.

Male rats received Gossypol 30 mg/kg body mass daily through intubation for 30 and 60 d. The testes were processed for histological and histochemical lipid staining, thiamine pyrophosphatase (TPPase), and alpha-glycerophosphate dehydrogenase (alpha-GPDH)--examinations. Gossypol induced effects were: various types of deformations of elongated spermatids, and their loss of orientation towards the lamina propria during the maturation phase; displacement of germ cells and occasionally also of Sertoli cells towards the lumen of the seminiferous tubule in form of a "puff". TPPase was observed in the supranuclear region of dislocated round spermatids and pachytene spermatocytes. alpha-GPDH was observed in the split pieces of mid- and tail pieces of elongated spermatids enclosed in the "puff". The induced effects were reversible.

In 40 male adult rats on unilateral torsion of the spermatic cord was initiated by operation. In the first group (4 x 5 animals) the testes were removed after 3, 6, 12, 24 hours. The treatment of the animals in the second group (4 x 5 animals) was untwisting of the torsion after 3, 6 12, 24 hours. Three months later both testes were removed. Histological and enzymehistochemical results are as follows: 1. The extend of acute damage to the affected testis depends on the length the torsion lasted. 2. The delay of time of untwisting the testis probably does not influence the degree of later regeneration. 3. Even short duration of torsion of the affected testis results in damage to the contralateral testies.

Biopsy material from infertility patients, suffering from varicocele orchiopathy as diagnosed by clinical and spermatological analysis, was processed for histological and enzyme-histochemical examinations.

Im Gegensatz zu den zahlreichen Veroffentlichungen uber die Resultate rein morphologischer, endokrinologischer und biochemischer Untersuchungen (Literaturtibersicht bei A. und E. Steinberger, 1980) liegen nur wenige Ergebnisse zur normalen und pathologisch veranderten Enzymzyto- und Enzymhistoarchitektur des menschlichen Hodens vor. So konnen z.B. durch enzymhistochemische Nachweise bestimmte Informationen uber die Entstehung und den Ablauf andrologischer Erkrankungen gewonnen werden; derartige Differenzierungen lassen sich mit rein histologischen Methoden nicht erfassen (Hofmann et al., 1980, 1982; Passia et al., 1980).

With the help of a modified dithizone method, heavy metals could be demonstrated in the sperms, the ejaculate fluid, and the spermatogenic cells of men. The heavy metals were localized in the sperms in form of small red granules particularly in the mid piece with a distribution like a "string of pearls". The sperms with a lower motility index often did not show any heavy metal granules in the mid pieces. The absence of essential heavy metals like zinc, iron or manganese may lead to the inhibition of enzyme systems in the mid pieces of the sperms, which are essential for the motility.

Calcium activated ATP-ase was studied enzymohistochemically in the testis of the common frog Rana temporaria during an annual reproduction cycle. The enzyme activity was localized in the follicle cells of the cyst wall, in the mature Sertoli cells, in germ cells and in the lamina propria of the seminiferous tubules. The maximum activity was recorded during the hibernation period, the cyst formation and the late maturation phase. The lowest activity was observed during the phases of spermiation and maturation of primary spermatocytes. The intensity of the ATP-ase reaction can be correlated with the seasonal changes during the spermatogenetic cycle of Rana temporaria. The role of ATP-ase in the permeability processes across the cyst wall, in the contractility of Sertoli cells and myoid cells of the lamina propria as well as in the release of sperms has been discussed.

Die perinatale Entwicklung der Leydigzellen im Rattentestis last sich in zwei deutlich voneinander abgrenzbare Zellpopulationen gliedern (Roosen-Runge und Anderson 1959; Lording und de Kretser 1972; Passia et al. 1979; Ziegler et al. 1983): 1. Die fetalen Leydigzellen, die um den 15. Fetaltag (f. d.) erscheinen, am 19. f. d. ein Maximum der 3β-Hydroxysteroid-Dehydrogenase (HSDH)-Aktivitat zeigen und bis zum 23. Postnatal (p. n.)-Tag morphologisch sichtbar sind. Diese Population wurde von Passia et al. (1979) aufgrund ihrer Lokalisation in den interstitiellen Dreiecken zwischen den Hodenkanalchen als intertubulare Leydigzellen (ILZ) bezeichnet. 2. Die postnatalen Leydigzellen, die am 13. Postnataltag erscheinen und auch den adulten Leydigzellen entsprechen; diese Zellen wurden von Passia et al. (1979) als peritubulare Leydigzellen (PLZ) bezeichnet. Die biochemischen Studien uber die Testosteronproduktion im Rattentestis wahrend der fetalen und postnatalen Entwicklung unterstutzen die oben erwahnten Befunde (Dohler und Wuttke 1974; Payne et al. 1980; Slob et al. 1980; Weisz und Ward 1980; Chase und Payne 1983).

The distribution of thiamine pyrophosphatase (TPPase) was studied in the human testicular biopsy tissue with the help of a modified histochemical gel method. The spermatogonia showed a large round strongly positive reaction zone in the supranuclear region. The pachytene spermatocytes exhibited a large semicircular TPPase reaction zone around the nucleus directing towards the lumen. The early round spermatids were characterized by a small, round, and weak supranuclear reaction zone. The elongated late spermatids were devoid of TPPase activity. The Sertoli cells possessed a "streamer" like formation of strong TPPase activity spreading from basal to apical portion.

In cases of varicocele, the clinical diagnosis is based on the nature of the scrotal varicosis and its associated characteristics, which may be as important in diagnosing this disease as the peculiar findings of the veins themselves. These characteristics are scrotal asthenia and low and horizontal position of the affected testicle, which is diminished in size and altered in consistency depending on the duration and degree of the vascular disturbance. Inguinal hernias, hemorrhoids, varicosis of the legs, and the overall leptosomatic constitution of the patients are further indications.

Ultrastructural studies on biopsy tissue from the right testis of a 39-year old patient with varicocele revealed 2.5-5 microns thick invaginations of the lamina propria towards the lumen of the seminiferous tubules. These invaginations were of various lengths. The presence of invaginations was confirmed through examination of serial semi-thin sections. In some seminiferous tubules two neighbouring deep invaginations were joined together thus completely encircling and thereby separating the basal compartment, and in some cases even the adluminal compartment, of the seminiferous tubule. The invaginations were surrounded continuously by the basement membrane and contained collagen fibres, cell processes of myoid cells and in some cases also their nuclei.

Summary The effects of an organosilicon compound SOC, a derivative of 1,3-dioxa-2-sila-cyclopentane, on rat spermiogenesis were studied with the help of histochemical and enzyme histochemical methods. The rats were injected with SOC i.p. daily for 3 weeks (dosage: 25 mg SOC/kg body weight). Two changes were observed: a) formation of a distinct vacuole within the acrosomal cap of spermatids b) the splitting of the tails of mature sperms.

Summary Zinc has been localized histochemically in the spermatotheka of the salamander Salamandra salamandra using a modified dithizone method. Results are discussed regarding the specifity of the method as well as the significance of Zn for long sperm storage and for possible reactivation of spermatozoa.